Ultraviolet B both damages the skin and is required for photochemical transformation of 7-dehydrocholesterol to vitamin D3 (D3). Its sequential hydroxylation at C25 and C1 generates biologically active 1,25(OH)2D3 that displays a variety of pleiotropic activities. It was believed that all of these effects are mediated by single molecule, 1,25(OH)2D3, and single receptor, VDR. Discovery of an alternative pathway in which CYP11A1 oxidizes the side chain of D3 producing 20(OH)D3 with its further metabolism to other downstream-derivatives, (OH)nD3, challenged this dogma. 20(OH)D3 is detectable in the human epidermis at concentration higher than 25(OH)D3, and in human serum at ~3nM. 20(OH)D3 shows biological activities suggestive that it can act as an endogenous regulator of epidermal barrier, while its presence in circulation suggests hormonal functions. 20(OH)D3 is noncalcemic at pharmacological doses (30-60g/kg). Initial data and computer modeling indicate that it can act as biased agonist on VDR and reverse agonist on ROR? and ROR?. Hypothesis: 20(OH)D3 and/or its metabolites acting directly on VDR and/or ROR? and ROR? stimulate keratinocyte differentiation program and photoprotective and repair mechanisms that protect epidermis against UVB-induced pathology. These effects would not require its hydroxylation in position C1?, in contrast to 1,25(OH)2D3. The hypothesis will be tested as follows: Aim 1. To test the relative role of VDR, ROR?, or ROR? in 20(OH)D3 mediated regulation of the proliferation and differentiation programs in epidermal keratinocytes. Subaim 1: Through in vitro binding to the ligand-binding domain of recombinant VDR and RORs we will define relative interactions of 20(OH)D3 and its downstream metabolites with the receptors in comparison to their native ligands. Then we will apply complex cell-based models to measure ligands modulated transcriptional activities. These will be supplemented by molecular modeling analyses. Subaim 2: The relative roles of VDR and RORs in regulation of keratinocytes proliferation and differentiation will be tested using gene silencing technology. This will be complemented by tests on keratinocytes isolated from VDR-/-, ROR?-/- or ROR?-/- mice. Subaim 3: RNA-Seq for gene expression and ChIP-Seq for receptor enrichment on target genes followed by bioinformatics analyses to identify alternative targets. Aim 2. To define protective role of 20(OH)D3 against UVB radiation in the human epidermis. Subaim 1: Testing the role of 20(OH)D3 as a survival factor. Subaim 2: Testing whether 20(OH)D3 attenuates UVB induced apoptosis. Subaim 3: Testing whether 20(OH)D3 can act as an antigenotoxic/antimutagenic agent. Subaim 4: Testing the role of ROR? or ROR? in skin responses to the UVB using mice with genotype -/-,-/+ and +/+ for these receptors and comparing with mice with defective or intact VDR. Aim 3. To evaluate whether the phenotypic effects of 20(OH)D3 and its metabolites require 1? hydroxylation as described for 1,25(OH)2D3. We will use cells with silenced CYP27B1 gene. This will be complemented by pharmacological inhibition of the CYP27B1 activity in keratinocytes.